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Cord blood stem cell use, cryopreservation and ethical dilemmas
Abstracts - WARM: In-vitro embryology: new trends in reproductive medicine
Israeli experience of ovarian tissue cryopreservation Abir R Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center, Petach Tikvah, Israel Chemotherapy and especially alkylating agents induce irreversible damage to the ovarian follicles as well as infertility. Human ovaries contain mainly primordial follicles that can be frozen successfully. Therefore, in various medical centres in Israel and abroad, ovarian tissue is cryopreserved from young female cancer patients preferably before chemotherapy. To date, ovarian tissue from 200 patients aged 6–40 years has been frozen in three major medical centres in Israel (Haddasah, Sheba and Rabin Medical Centres). Most of the patients had bone sarcomas, Hodgkin lymphoma and leukaemias, but breast cancer and other malignancies also occurred within this patient group. Fertility restoration will be achieved in the future by either transplantation of the ovarian tissue or, further into the future, by in-vitro maturation (IVM) of primordial follicles, followed by routine IVF and embryo transfer. The later method will eliminate the possible risk of malignancy transmission via grafts, especially in cases of leukaemias, non-Hodgkin’s lymphoma and breast cancer. Recently, a live birth has been reported from Sheba Medical Centre after transplantation of frozen–thawed ovarian strips followed by IVF. Unfortunately, in some cases ovarian cryopreservation can be conducted only after initiation of chemotherapy. Therefore, in a study conducted in the laboratory, preliminary results indicate that in these cases, cryopreservation can be considered only in patients aged not more than about 20 years. Although all medical centres in Israel use slow-gradual ovarian fragment freezing with a dimethylsulfoxide or an ethylene glycol protocol, cryopreservation of whole ovine ovaries with their pedicle followed by vascular surgery is being investigated at the Agriculture Research Organization. The possible advantage of this method is rapid vascularization of the grafts. At the Hadassah Medical Centre, cryopreservation of ovarian tissue is accompanied by cryopreservation of immature oocytes aspired from antral follicles, cryopreservation of the IVM oocytes or cryopreservation of embryos derived from the IVM oocytes. However, in most cases very few antral follicles can be detected in the ovaries, especially in samples from premenarcheal girls or when a full oophorectomy is not performed but a limited portion of the ovary is retrieved. In conclusion, in recent years there is an increase in awareness amongst clinicians and patients of the risk of chemotherapy to fertility. Moreover, the issue of having children is crucial for both the Jewish and Moslem populations in Israel. Especially after the birth of the Israeli baby from grafted frozen–thawed ovarian tissue, an increasing number of cancer patients are undergoing ovarian cryopreservation, or choose this option for children born subsequent to the first ones. Comparison between oocyte slow and fast freezing Chen ZJ Reproductive Medical Research Center, Shandong Provincial Hospital, Shandong University, Reproductive Medical Research Building, 324 Jingwu Road, 250100 Jinan, China In humans, the main application of oocyte cryopreservation technique is the possibility of fertility restoration, by facilitating oocyte donation and storing the excess of oocytes during the assisted reproductive treatment therapies. Slow freezing is a
2
fairly active area of investigation for human oocytes with much data from oocyte slow-freezing protocols having been reported. Despite much improvement, no more than 200 babies have been born by means of slow freezing the oocyte. Lower survival rate and lower quality of embryo are the major problems that have blocked the clinical application of oocyte cryopreservation. Though the idea of vitrifying living systems was raised around 70 years ago by Luyet, successful pregnancies and deliveries after vitrification and warming of human oocytes were not reported until 1999. In the past 2 years, oocyte vitrification has been introduced with great progress. Slow freezing means attempting to keep equilibrium between the various sources of injury, with low cryoprotectant concentration and ice formation control, which results in the vitrification of solutes in and close to the cell. The most commonly used slow freezing method is in the presence of 1.5 mol propanediol plus sucrose. 0.25 ml plastic straws and an automated programme freezer are also required. The improvement in oocyte slow freezing methodology came with the determination of optimal sucrose concentrations to use in the freezing medium. Protocols using slow freezing were further fine-tuned by varying exposure time and temperature to cryoprotectant solutions before and after freezing and on Na-replacement with choline, leading to increased survival. Vitrification is the direct conversion of liquid state into glass by specific condition of ultra-fast cooling and solute concentration that inhibit ice nucleation and growth, bypassing critical temperature of ice formation (no phase transition). Successful vitrification requires minimum volume of holding media, increased viscosity, higher cryoprotectant concentration, high cooling and warming rates, and special vehicle device (carrier). Various carriers (electron microscope grid, open-pulled straws, Flexipet-denuding pipette hemistraws, cryoloop, Cryotop) have been introduced since 1996. The use of decreased cryoprotectant concentration, together with rapid loading and expelling, minimizes the toxic and osmotic injury. There are many disadvantages to slow freezing methods, including no control over dehydration rate, no maintenance of physiological temperature during equilibration, prolonged temperature shock, possible fracture of zona pellucida, ice crystallization, expensive running costs as well as the time needed. Vitrification is a simple and a promising technique for cryopreservation. Its advantages are less mechanical injury (extracellular crystal formation), less osmotic stress, no intracellular crystal formation, less labour and time, simple protocol and no expensive device needed. Though vitrification is competitive to slow freezing, many disadvantages should be pointed out, such as higher cryoprotectant concentration and direct contact with liquid nitrogen by the carriers. It is difficult to maintain the time and temperature required for high speed cooling. Skilled and quick operation should be performed before a more efficient technique is invented. Further study should be carried out using less toxic and well-tolerated cryoprotectants at low concentration, considering pre-freeze temperature and time of exposure. Ultrastructure study of oocytes should be addressed to investigate the effect of vitrification procedure on the subcellular organelles, cytoskeleton, meiotic spindle and other microstructures. Cord blood stem cell use, cryopreservation and ethical dilemmas Feldberg D Helen Schneider Hospital for Women, Rabin Medical Centre and Tel-Aviv University School of Medicine, Israel
Abstracts - WARM: In-vitro embryology: new trends in reproductive medicine
The blood that remains in the placenta after birth is readily available, can be collected at no risk to the mother or newborn and may be stored frozen for years. Cord blood has less restrictive HLA-compatibility requirements than bone marrow and can be provided quickly. Umbilical cord blood is increasingly used as a source of stem cells to repopulate the bone marrow in the treatment of life threatening diseases in children and adults. Although exact statistics are not available, about 6000 cord blood transplantations have now been performed worldwide, primarily in the USA, Western Europe, Japan, Australia and Israel. The number of transplantations performed annually is increasing and adults now account for about one-third of all recipients. About two-thirds of transplants are used in patients with leukaemia (adults and children) and about one-quarter in patients and children with genetic diseases. This scientific progress, however, has triggered a continuous debate about how to organize cord blood banks and the role of public and private facilities. In this presentation, the scientific basis, the therapeutic approach and enormous potential of the cord blood stem cell use in oncological and regenerative medicine, including the ethical and moral aspects, will be thoroughly analysed. Practical and scientific perspectives of cryoprotectant-free vitrification of human spermatozoa Isachenko E Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany It is well known that the use of permeable cryoprotectants for the conventional cryopreservation of human spermatozoa, oocytes and different stages of embryos including blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high concentrations of permeable cryoprotectants (30–50%) with the consequent cytotoxic effects due to osmotic stress and cannot be applied to sperm cryopreservation due to lethal effect of osmotic shock on the spermatozoa. However, the human spermatozoa can be successfully cryopreserved avoiding the use of permeable cryoprotectants through vitrification at very high cooling rates (up to 7.2 × 105°C/min). Herein, the history of this problem is reviewed, and in this light offer an explanation through physico-chemical concepts for one of the most recent developments in this area: the recovery of the motile and viable spermatozoa after cryoprotectant-free vitrification. Slow freezing versus vitrification: techniques and results of human oocytes and embryos Al-Hasani S Women Hospital, University of Schleswig-Holstein, Campus Lübeck, Germany Nine months before the first successful pregnancy from IVF, at a CIBA Foundation symposium in London in January 1977, Robert Edwards stated: ‘The storage of human preimplantation embryos at lower temperature could be valuable in clinical practice for the cure of infertility and possibly to avert inherited defects in children’. The same author suggested that it might be beneficial to cryopreserve human oocytes. Since the birth of Louise Brown in 1978, more than one million children have been born as a result of assisted reproductive treatments. Although difficult to document specific numbers, it
has been estimated that tens of thousands of children were born from embryos that had been cryopreserved. In 1985, ice-free cryopreservation of mouse embryos at –196°C by vitrification was reported as an alternative approach to traditional slowcooling/rapid-thaw protocols and vitrification techniques have entered the mainstream of animal reproduction more and more. In addition, the last few years have seen a significant revival of interest in the potential benefits of vitrification protocols and techniques in human assisted reproductive treatments. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then to be plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human assisted reproduction. This may be due to the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. The different vitrification carrier solutions, as well as new research efforts, will be discussed. Vitrification of human oocytes Antinori, S, Licata, E, Antinori, M, Dani, G, Cerusico, F, Versaci C RAPRUI Day-Hospital International Associated Research Institute for Human Reproduction Infertility Unit, Rome, Italy Introduction: The vitrification of oocytes and embryos is widely used nowadays. The conventional methods of ‘slow and fast’ freezing have offered and will offer optimal results, but at present attention is paid to the vitrification procedure which implies a higher concentration of cryoprotectants and less time to perform it. This method avoids any possible damage, since oocytes and embryos are put directly in liquid nitrogen and the formation of ice crystals is prevented. The cryoprotectants can be dangerous in high concentrations: therefore different protocols have been studied with a view to optimizing and simplifying these procedures. Ethylene glycol was employed as cryoprotectant and a high rate of oocyte survival has been achieved. Materials and methods: The oocytes are put on a Petri dish containing only equilibration solution (7.5%, Ethylene Glycol; 7.5% DMSO) for 5–15 min at room temperature. Soon after, oocytes are aspirated, and plunged into the vitrification solution (15% Ethylene Glycol; 15% DMSO; 0.5 M Sucrose) for about 60 sec at room temperature. After having observed that cellular shrinkage has taken place, oocytes are aspirated and put on the top of a cryotop. Each cryotop can store not more than three oocytes. The cryotop is then put into liquid nitrogen and stored. The warming of oocytes is performed by putting the cryotop in thawing solution (1M Sucrose) for 60 s at 37°C. Oocytes are plunged into 0.5 M sucrose for 3 min. The cryopreserving agents are removed by washing the oocytes into isotonic solution twice for 5 min (Ham’s F-10 + 20% serum). Then they are transferred into the incubator for 2 h before performing intracytoplasmic sperm injection (ICSI). On the day 21 of the cycle, the patient is administered GnRH
3
Israeli experience of ovarian tissue cryopreservation Abir R Infertility and IVF Unit, Helen Schneider Hospital for Women, Rabin Medical Center, Petach Tikvah, Israel Chemotherapy and especially alkylating agents induce irreversible damage to the ovarian follicles as well as infertility. Human ovaries contain mainly primordial follicles that can be frozen successfully. Therefore, in various medical centres in Israel and abroad, ovarian tissue is cryopreserved from young female cancer patients preferably before chemotherapy. To date, ovarian tissue from 200 patients aged 6–40 years has been frozen in three major medical centres in Israel (Haddasah, Sheba and Rabin Medical Centres). Most of the patients had bone sarcomas, Hodgkin lymphoma and leukaemias, but breast cancer and other malignancies also occurred within this patient group. Fertility restoration will be achieved in the future by either transplantation of the ovarian tissue or, further into the future, by in-vitro maturation (IVM) of primordial follicles, followed by routine IVF and embryo transfer. The later method will eliminate the possible risk of malignancy transmission via grafts, especially in cases of leukaemias, non-Hodgkin’s lymphoma and breast cancer. Recently, a live birth has been reported from Sheba Medical Centre after transplantation of frozen–thawed ovarian strips followed by IVF. Unfortunately, in some cases ovarian cryopreservation can be conducted only after initiation of chemotherapy. Therefore, in a study conducted in the laboratory, preliminary results indicate that in these cases, cryopreservation can be considered only in patients aged not more than about 20 years. Although all medical centres in Israel use slow-gradual ovarian fragment freezing with a dimethylsulfoxide or an ethylene glycol protocol, cryopreservation of whole ovine ovaries with their pedicle followed by vascular surgery is being investigated at the Agriculture Research Organization. The possible advantage of this method is rapid vascularization of the grafts. At the Hadassah Medical Centre, cryopreservation of ovarian tissue is accompanied by cryopreservation of immature oocytes aspired from antral follicles, cryopreservation of the IVM oocytes or cryopreservation of embryos derived from the IVM oocytes. However, in most cases very few antral follicles can be detected in the ovaries, especially in samples from premenarcheal girls or when a full oophorectomy is not performed but a limited portion of the ovary is retrieved. In conclusion, in recent years there is an increase in awareness amongst clinicians and patients of the risk of chemotherapy to fertility. Moreover, the issue of having children is crucial for both the Jewish and Moslem populations in Israel. Especially after the birth of the Israeli baby from grafted frozen–thawed ovarian tissue, an increasing number of cancer patients are undergoing ovarian cryopreservation, or choose this option for children born subsequent to the first ones. Comparison between oocyte slow and fast freezing Chen ZJ Reproductive Medical Research Center, Shandong Provincial Hospital, Shandong University, Reproductive Medical Research Building, 324 Jingwu Road, 250100 Jinan, China In humans, the main application of oocyte cryopreservation technique is the possibility of fertility restoration, by facilitating oocyte donation and storing the excess of oocytes during the assisted reproductive treatment therapies. Slow freezing is a
2
fairly active area of investigation for human oocytes with much data from oocyte slow-freezing protocols having been reported. Despite much improvement, no more than 200 babies have been born by means of slow freezing the oocyte. Lower survival rate and lower quality of embryo are the major problems that have blocked the clinical application of oocyte cryopreservation. Though the idea of vitrifying living systems was raised around 70 years ago by Luyet, successful pregnancies and deliveries after vitrification and warming of human oocytes were not reported until 1999. In the past 2 years, oocyte vitrification has been introduced with great progress. Slow freezing means attempting to keep equilibrium between the various sources of injury, with low cryoprotectant concentration and ice formation control, which results in the vitrification of solutes in and close to the cell. The most commonly used slow freezing method is in the presence of 1.5 mol propanediol plus sucrose. 0.25 ml plastic straws and an automated programme freezer are also required. The improvement in oocyte slow freezing methodology came with the determination of optimal sucrose concentrations to use in the freezing medium. Protocols using slow freezing were further fine-tuned by varying exposure time and temperature to cryoprotectant solutions before and after freezing and on Na-replacement with choline, leading to increased survival. Vitrification is the direct conversion of liquid state into glass by specific condition of ultra-fast cooling and solute concentration that inhibit ice nucleation and growth, bypassing critical temperature of ice formation (no phase transition). Successful vitrification requires minimum volume of holding media, increased viscosity, higher cryoprotectant concentration, high cooling and warming rates, and special vehicle device (carrier). Various carriers (electron microscope grid, open-pulled straws, Flexipet-denuding pipette hemistraws, cryoloop, Cryotop) have been introduced since 1996. The use of decreased cryoprotectant concentration, together with rapid loading and expelling, minimizes the toxic and osmotic injury. There are many disadvantages to slow freezing methods, including no control over dehydration rate, no maintenance of physiological temperature during equilibration, prolonged temperature shock, possible fracture of zona pellucida, ice crystallization, expensive running costs as well as the time needed. Vitrification is a simple and a promising technique for cryopreservation. Its advantages are less mechanical injury (extracellular crystal formation), less osmotic stress, no intracellular crystal formation, less labour and time, simple protocol and no expensive device needed. Though vitrification is competitive to slow freezing, many disadvantages should be pointed out, such as higher cryoprotectant concentration and direct contact with liquid nitrogen by the carriers. It is difficult to maintain the time and temperature required for high speed cooling. Skilled and quick operation should be performed before a more efficient technique is invented. Further study should be carried out using less toxic and well-tolerated cryoprotectants at low concentration, considering pre-freeze temperature and time of exposure. Ultrastructure study of oocytes should be addressed to investigate the effect of vitrification procedure on the subcellular organelles, cytoskeleton, meiotic spindle and other microstructures. Cord blood stem cell use, cryopreservation and ethical dilemmas Feldberg D Helen Schneider Hospital for Women, Rabin Medical Centre and Tel-Aviv University School of Medicine, Israel
Abstracts - WARM: In-vitro embryology: new trends in reproductive medicine
The blood that remains in the placenta after birth is readily available, can be collected at no risk to the mother or newborn and may be stored frozen for years. Cord blood has less restrictive HLA-compatibility requirements than bone marrow and can be provided quickly. Umbilical cord blood is increasingly used as a source of stem cells to repopulate the bone marrow in the treatment of life threatening diseases in children and adults. Although exact statistics are not available, about 6000 cord blood transplantations have now been performed worldwide, primarily in the USA, Western Europe, Japan, Australia and Israel. The number of transplantations performed annually is increasing and adults now account for about one-third of all recipients. About two-thirds of transplants are used in patients with leukaemia (adults and children) and about one-quarter in patients and children with genetic diseases. This scientific progress, however, has triggered a continuous debate about how to organize cord blood banks and the role of public and private facilities. In this presentation, the scientific basis, the therapeutic approach and enormous potential of the cord blood stem cell use in oncological and regenerative medicine, including the ethical and moral aspects, will be thoroughly analysed. Practical and scientific perspectives of cryoprotectant-free vitrification of human spermatozoa Isachenko E Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany It is well known that the use of permeable cryoprotectants for the conventional cryopreservation of human spermatozoa, oocytes and different stages of embryos including blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high concentrations of permeable cryoprotectants (30–50%) with the consequent cytotoxic effects due to osmotic stress and cannot be applied to sperm cryopreservation due to lethal effect of osmotic shock on the spermatozoa. However, the human spermatozoa can be successfully cryopreserved avoiding the use of permeable cryoprotectants through vitrification at very high cooling rates (up to 7.2 × 105°C/min). Herein, the history of this problem is reviewed, and in this light offer an explanation through physico-chemical concepts for one of the most recent developments in this area: the recovery of the motile and viable spermatozoa after cryoprotectant-free vitrification. Slow freezing versus vitrification: techniques and results of human oocytes and embryos Al-Hasani S Women Hospital, University of Schleswig-Holstein, Campus Lübeck, Germany Nine months before the first successful pregnancy from IVF, at a CIBA Foundation symposium in London in January 1977, Robert Edwards stated: ‘The storage of human preimplantation embryos at lower temperature could be valuable in clinical practice for the cure of infertility and possibly to avert inherited defects in children’. The same author suggested that it might be beneficial to cryopreserve human oocytes. Since the birth of Louise Brown in 1978, more than one million children have been born as a result of assisted reproductive treatments. Although difficult to document specific numbers, it
has been estimated that tens of thousands of children were born from embryos that had been cryopreserved. In 1985, ice-free cryopreservation of mouse embryos at –196°C by vitrification was reported as an alternative approach to traditional slowcooling/rapid-thaw protocols and vitrification techniques have entered the mainstream of animal reproduction more and more. In addition, the last few years have seen a significant revival of interest in the potential benefits of vitrification protocols and techniques in human assisted reproductive treatments. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then to be plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human assisted reproduction. This may be due to the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. The different vitrification carrier solutions, as well as new research efforts, will be discussed. Vitrification of human oocytes Antinori, S, Licata, E, Antinori, M, Dani, G, Cerusico, F, Versaci C RAPRUI Day-Hospital International Associated Research Institute for Human Reproduction Infertility Unit, Rome, Italy Introduction: The vitrification of oocytes and embryos is widely used nowadays. The conventional methods of ‘slow and fast’ freezing have offered and will offer optimal results, but at present attention is paid to the vitrification procedure which implies a higher concentration of cryoprotectants and less time to perform it. This method avoids any possible damage, since oocytes and embryos are put directly in liquid nitrogen and the formation of ice crystals is prevented. The cryoprotectants can be dangerous in high concentrations: therefore different protocols have been studied with a view to optimizing and simplifying these procedures. Ethylene glycol was employed as cryoprotectant and a high rate of oocyte survival has been achieved. Materials and methods: The oocytes are put on a Petri dish containing only equilibration solution (7.5%, Ethylene Glycol; 7.5% DMSO) for 5–15 min at room temperature. Soon after, oocytes are aspirated, and plunged into the vitrification solution (15% Ethylene Glycol; 15% DMSO; 0.5 M Sucrose) for about 60 sec at room temperature. After having observed that cellular shrinkage has taken place, oocytes are aspirated and put on the top of a cryotop. Each cryotop can store not more than three oocytes. The cryotop is then put into liquid nitrogen and stored. The warming of oocytes is performed by putting the cryotop in thawing solution (1M Sucrose) for 60 s at 37°C. Oocytes are plunged into 0.5 M sucrose for 3 min. The cryopreserving agents are removed by washing the oocytes into isotonic solution twice for 5 min (Ham’s F-10 + 20% serum). Then they are transferred into the incubator for 2 h before performing intracytoplasmic sperm injection (ICSI). On the day 21 of the cycle, the patient is administered GnRH
3